Shimomura K

Tumour-amplified kinase BTAK is amplified and overexpressed in gastric cancers with possible involvement in aneuploid formation

Our recent analysis of gastric cancers using comparative genomic hybridization (CGH) revealed a novel high frequent copy number increase in the long arm of chromosome 20. Tumour-amplified kinase BTAK was recently cloned from breast cancers and mapped on 20q13 as a target gene for this amplification in human breast cancers. In the study presented here, we analysed BTAK copy-number and expression, and their relation to the ploidy pattern in 72 primary gastric cancers. Furthermore, wild-type BTAK and its deletion mutants were transfected to gastric cancers to examine changes in cell proliferation and DNA ploidy pattern. Evaluation of 72 unselected primary gastric cancers found BTAK amplification in 5% and overexpression in more than 50%. All four clinical samples with BTAK amplification showed aneuploidy and poor prognosis. Transfection of BTAK in near-diploid gastric cancers induced another aneuploid cell population. In contrast, the c-terminal-deleted mutant of BTAK induced no effect in DNA ploidy pattern and inhibited gastric cancer cell proliferation. These results suggest that BTAK may be involved in gastric cancer cell aneuploid formation, and is a candidate gene for the increase in the number of copies of the 20q, and thus may contribute to an increase in the malignant phenotype of gastric cancer.

Differential gene expression profiles of gastric cancer cells established from primary tumour and malignant ascites

Advanced gastric cancer is often accompanied by metastasis to the peritoneum, resulting in a high mortality rate. Mechanisms involved in gastric cancer metastasis have not been fully clarified because metastasis involves multiple steps and requires a combination of altered expressions of many different genes. Thus, independent analysis of any single gene would be insufficient to understand all of the aspects of gastric cancer peritoneal dissemination. In this study, we performed a global analysis of the differential gene expression of a gastric cancer cell line established from a primary main tumour (SNU-1) and of other cell lines established from the metastasis to the peritoneal cavity (SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB). The application of a high-density cDNA microarray method made it possible to analyse the expression of approximately 21 168 genes. Our examinations of SNU-5, SNU-16, SNU-620, KATO-III and GT3TKB showed that 24 genes were up-regulated and 17 genes down-regulated besides expression sequence tags. The analysis revealed the following altered expression such as: (a) up-regulation of CD44 (cell adhesion), keratins 7, 8, and 14 (epitherial marker), aldehyde dehydrogenase (drug metabolism), CD9 and IP3 receptor type3 (signal transduction); (b) down-regulation of IL2 receptor γ, IL4-Stat (immune response), p27 (cell cycle) and integrin β4 (adhesion) in gastric cancer cells from malignant ascites. We then analysed eight gastric cancer cell lines with Northern blot and observed preferential up-regulation and down-regulation of these selected genes in cells prone to peritoneal dissemination. Reverse transcriptase–polymerase chain reaction confirmed that several genes selected by DNA microarray were also overexpressed in clinical samples of malignant ascites. It is therefore considered that these genes may be related to the peritoneal dissemination of gastric cancers. The results of this global gene expression analysis of gastric cancer cells with peritoneal dissemination, promise to provide a new insight into the study of human gastric cancer peritoneal dissemination.

Overexpression of dopa decarboxylase in peritoneal dissemination of gastric cancer and its potential as a novel marker for the detection of peritoneal micrometastases with real-time RT–PCR

We previously performed a global analysis of the gene expression of gastric cancer cell lines established from metastases to the peritoneal cavity with the cDNA microarray method, which made it possible to analyse the expression of approximately 21 168 genes for the identification of novel markers for the detection of micrometastases in the peritoneal cavity. One of the upregulated genes is dopa decarboxylase (DDC), which is responsible for the synthesis of the key neurotransmitters dopamine and serotonine. We have examined its potential as a novel marker for the detection of peritoneal micrometastases of gastric cancer.DDC mRNA in the peritoneal wash from 112 gastric cancer patients was quantified for comparison of carcinoembryonic antigen (CEA) mRNA by means of real-time reverse transcriptase–polymerase chain reaction (RT–PCR) with a fluorescently labelled probe to predict peritoneal recurrence. The quantity of DDC and CEA correlated with wall penetration. Real-time RT–PCR could quantitate 10–106 DDC-expressing gastric cancer cells per 107 mesothelial cells. The cutoff value was set at the upper limit of the quantitative value for noncancer patients, and those above this cutoff value constituted the micrometastasis (MM+) group. Of 15 cases with peritoneal dissemination, 13 were MM+DDC (87% sensitivity), and one of 48 t1 cases was MM+ (98% specificity). DDC levels in peritoneal washes from patients with synchronous peritoneal metastases were more than 50 times higher than in those from patients without metastasis (P<0.01). For 15 cases of peritoneal dissemination (seven cases were cytologically positive), DDC was positive in 13 cases (87% sensitivity), but CEA failed to detect micrometastases in four cases (73% sensitivity), indicating that DDC is in some cases superior to CEA for the detection of peritoneal micrometastases of gastric cancer in terms of sensitivity as well as specificity, especially for poorly differentiated adenocarcinomas. A combination of CEA and DDC improved the accuracy of diagnosis up to 94%.These results suggest that DDC is potentially a novel marker for peritoneal dissemination of gastric cancer and that quantitative RT–PCR of DDC is reliable and efficient for the selection of patients for adjuvant intraperitoneal chemotherapy to prevent peritoneal recurrence.

Frequent loss of RUNX3 gene expression in remnant stomach cancer and adjacent mucosa with special reference to topography

Our previous studies suggest that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer. This study was conducted to determine whether alteration of RUNX3 gene expression could be detected in the normal-looking gastric remnant mucosa, and to ascertain any difference in the potential of gastric carcinogenesis between the anastomotic site and other areas in the remnant stomach after distal gastrectomy for peptic ulcer (RB group) or gastric cancer (RM group), by analysing RUNX3 expression with special reference to topography. A total of 89 patients underwent distal gastrectomy for gastric cancer from the intact stomach (GCI group) and 58 patients underwent resection of the remnant stomach for gastric cancer (RB group: 34 cases, RM group: 24 cases). We detected RUNX3 and gene promoter methylation by in situ hybridisation, quantitative reverse transcriptase–polymerase chain reaction (RT–PCR), and methylation-specific PCR. The interval between the initial surgery and surgery for remnant gastric cancer (interval time) was 10.4 years in the RM group, and 27.5 years in the RB group. Cancers in the RB group were significantly more predominant in the anastomosis area (P<0.05). Within the tumour, downregulation of RUNX3 expression ranged from 74.7 to 85.7% in the three groups. The rate of downregulation of RUNX3 of adjacent mucosa was 39.2% (11 in 28 cases) in RB and 47.6% (10 in 21 cases) in RM, which are significantly higher than that of the GCI group (19.5%, 17 in 87 cases). In noncancerous mucosa of the remnant stomach in the RB group, RUNX3 expression decreased more near the anastomosis area. In the RM group, however, there were no significant differences in RUNX3 expression by sampling location. Based on RUNX3 downregulation and clinical features, residual stomach mucosa of the RM group would have a higher potential of gastric carcinogenesis compared to the RB or GCI group. Gastric stump mucosa of the RB group has higher potential especially than other areas of residual stomach mucosa. Measurement of RUNX3 expression and detection of RUNX3 methylation in remnant gastric mucosa may estimate the forward risk of carcinogenesis in the remnant stomach.

Mutations in Exon 11 of the c-kit Gene in a Myogenic Tumor and a Neurogenic Tumor as Well as in Gastrointestinal Stromal Tumors

Background/Aims: Gain-of-function mutations in exons 9, 11 and 13 of the c-kit gene in gastrointestinal stromal tumors (GISTs) have been identified, and it has been reported that the prognosis is worse for patients with mutation-positive GISTs than for those with mutation-negative GISTs. We studied c-kit mutations in gastrointestinal mesenchymal tumors. By chance, the c-kit mutation in exon 11 was found in myogenic and neurogenic tumors as well as in GISTs. Furthermore, we studied the clinical prognostic utility of these mutations. Methods: Ten gastrointestinal mesenchymal tumors were stained with HE and immunohistochemically analyzed with α-smooth muscle actin, S-100 protein, CD34 and c-kit. In these tumors, as well as in 11 cases of leiomyomas, PCR-amplified DNA from the juxtamembrane (JM) domain of exon 11, the extracellular domain of exon 9 and the tyrosine kinase domain 1 of exon 13 showed a high frequency of c-kit mutation and was sequenced. Results: Although c-kit mutations have previously been reported only in GISTs, we found c-kit mutations in the JM domain of exon 11 in one myogenic and one neurogenic tumor as well as in two GISTs. No c-kit mutation was seen in the 11 cases of leiomyomas. In addition, all four cases with c-kit mutation in exon 11 suffered a relapse sooner than the other cases without c-kit mutations. Conclusion: Clinically, the prognosis was worse for the patients with mutation-positive gastrointestinal mesenchymal tumors than for those with mutation-negative tumors. We therefore conclude that the gain-of-function mutation in exon 11 of the c-kit gene is an important prognostic factor for gastrointestinal mesenchymal tumors, including myogenic and neurogenic tumors as well as GISTs.

Polymerase chain reaction for detection of carcinoembryonic antigen-expressing tumor cells on milky spots of the greater omentum in gastric cancer patients: a pilot study.

Our recent studies indicate that omental milky spots are frequently involved in the early stage of peritoneal cancer dissemination. We have used carcinoembryonic antigen (CEA)‐specific RT‐PCR for omental milky spots to predict peritoneal recurrence in gastric cancer patients. CEA mRNA was found to be positive in both 10 peritoneal washes and 16 greater omenta of 30 gastric cancer patients, including all 6 patients who showed positive results for both cytology and RT‐PCR of peritoneal wash and omentum. Three of the 6 cases with positive RT‐PCR in the greater omentum but not in the peritoneal wash showed recurrence of peritoneal carcinomatosa within 2 years after operation. Micrometastasis on omental milky spots was histologically confirmed in 6 of 30 gastric cancer cases. Non‐specific band was detected only in the omentum of 1 case of 15 benign disease (7%), but not in peritoneal washes (0%), probably due to weak expression of CEA in mesothelial cells. Our results show that CEA‐specific RT‐PCR targeting micro‐metastases on omental milky spots is more sensitive than targeting the peritoneal wash or conventional cytology, and suggest that this method is useful for the prediction of peritoneal recurrence in gastric cancer patients.